The present invention relates to novel phenylalanine ammonia-lyase-producing microorganisms and a method for their selection and use. More particularly, the invention concerns microorganisms which produce relatively high levels of the enzyme, phenylalanine ammonia-lyase (hereinafter referred to as PAL) in the absence of known inducers. PAL, in turn, is useful for the production of L-phenylalanine.
L-phenylalanine is an essential amino acid in man and is, therefore, an important ingredient of enteral and parenteral nutritional formulations. In addition, this amino acid is useful as a starting material for the production of other products, such as the artificial sweetener, aspartame. Various microbial processes for the production of phenylalanine are known. For example, U.S. Pat. No. 3,660,235 describes the production of phenylalanine by phenylalanine analog resistant strains of Brevibacterium, Corynebacterium, Arthrobacter, Bacillus, and Candida. The production of this amino acid by tyrosine-requiring mutants of certain strains of Brevibacterium, Corynebacterium, Arthrobacter, and Escherichia is also known. See U.S. Pat. Nos. 3,654,079 and 3,909,353.
PAL catalyzes the breakdown of L-phenylalanine to trans-cinnamic acid and ammonia. This enzymatic reaction is reversible, and British Patent No. 1,489,468 discloses a process for the production of L-phenylalanine which involves the PAL-catalyzed reaction of trans-cinnamic acid with ammonium ions to yield L-phenylalanine. This reaction is useful for producing L-phenylalanine and, therefore, there is a continuing need to obtain production microorganisms which produce high levels of PAL activity. Such microorganisms can be used directly for the conversion of cinnamic acid and ammonium ions to L-phenylalanine, or the enzyme can be isolated from the cells and used to produce L-phenylalanine in various forms of bioreactors.
Microbial biosynthesis of PAL is highly regulated and current production strains require the use of an inducer to elicit PAL synthesis. This inducer, usually L-phenylalanine, contributes to the total raw material costs of PAL fermentation. Therefore, the commercial need for a microorganism that can synthesize PAL in the absence of an inducer persists.